Method And Apparatus For Continuous Removal Of Submicron Sized Particles In A Closed Loop Liquid Flow System

ABSTRACT

A method and apparatus for continuous removal of submicron sized artificial oxygen carriers (rAOC) and other materials such as cancer cells and bacteria from blood and other liquids. A centrifuge rotor having a curved shape is offset on a spinning rotor base and creates contiguous areas of low to high centrifugal force depending on the distances from the axis of the rotor base. This creates a density gradient field that separates materials of different densities input to the centrifuge that exit via different outputs. A monitor detects any red blood cells (RBC) with the rAOC before they exit the centrifuge. If there are any RBC detected logic circuitry changes the speed of rotation of the rotor, and the flow rate of pumps inputting and removing separated blood and rAOC to and from the centrifuge until there are no RBC in the rAOC exiting the centrifuge.

This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Application No. 61/236,810 , filed on Aug. 25, 2009.

Field of the Invention

The present invention relates to a method and apparatus for continuousremoval of submicron sized particles from blood or other liquids.

BACKGROUND OF THE INVENTION

In the prior art there are a range of particulate carriers intended forthe controlled delivery of biologically active substances within thebody. Their sizes range from micron to submicron, and their compositionsrange from organic (e.g. polymers, lipids, surfactants, proteins) toinorganic (calcium phosphate, silicate, CdSe, CdS, ZnSe, gold andothers). Each of these particulate carriers are designed to carry achemically or biochemically reactive substance, and either release itover time, or at a specific location, or both.

The size of the individual particulate carriers and their load capacityis controlled by the amount of material used in the synthesis, themorphology by which the components assemble, and the specificcomposition of the components. The synthesized particulate carriers havethe dual function of being able to solubilize or to be able to bind tothe chemically or biochemically reactive substances intended forultimate delivery. The underlying assumption is that the enclosedreactive substances will ultimately be released so that they can performtheir intended functions. The particulate carriers themselves usually donot participate in the release function, except to the extent that theyregulate the timing or location of release of the reactive substancesthey carry, and the carrier components must either decompose over time,or remain as non-active and non-toxic substances that do not cause anyharm.

In the field of medicine such particulate carriers have been used toserve as artificial oxygen carriers (AOC) in artificial blood products.Artificial blood is a product made to act as a substitute for red bloodcells which transport oxygen and carbon dioxide throughout the body.However, the function of real blood is complicated, and the developmentof artificial blood has generally focussed on meeting only a specificfunction, gas exchange - oxygen and carbon dioxide.

Whole blood serves many different functions that cannot be duplicated byan AOC. Artificial blood mixable with autologous blood can supportpatients during surgery and support transfusion services in emergingcountries with limited healthcare, blood donations and storagefacilities, or high risk of exposure to disease since screeningprocedures are too expensive. An AOC is a blood substitute, which is notdependent upon cross matching and blood-typing would mean no delay inblood availability, and could mean the difference between life and deathof patients. In prior art medical applications the residual materialsfrom particulate carriers are expected to be metabolized and/or excretedover time. However, the disposal of particulate carriers with naturalmetabolism of the patients is extremely difficult.

Another motivation for developing improved AOC is that despitesignificant advances in donated blood screening there are still concernsover the limited shelf life which is 42 days at 2°-6° C.

In the era of modern science, several decades of extensive academic,industry research efforts, clinical trials, and spending multiplebillions of dollars, has led to two major classes of AOCs, namelyemulsified perfluorocarbons (PFC) and polymeric hemoglobins (Hb). Whilethese two types of AOCs each have some advantages, none are yet approvedfor clinical use in the U.S.

Chemically and biologically inert, emulsified, sterilizedperfluorocarbons (PFCs) are stable in storage at low temperatures 2-5°C. for over a year. Further, PFCs are relatively inexpensive to produceand can be made devoid of any biological materials eliminating thepossibility of spreading an infectious disease via a blood transfusion.Because they are not soluble in water they must be combined withemulsifiers able to suspend tiny droplets of PFC in the blood. In vivothe perfluorocarbon is ultimately expelled via the lungs after digestionof the emulsifier by the macrophage/monocyte system. In addition, PFCsare biologically inert materials that can dissolve about fifty timesmore oxygen than blood plasma but less oxygen than red blood cells. Forinstance, a mixture consisting of 70% blood and 30% perfluorocarbon byvolume can provide the needed 5 ml of oxygen per 100 ml of blood if thepartial pressure of oxygen in the lungs can be increased to 120 mm Hg byhaving the patient breath air with an oxygen partial pressure ofapproximately 180 mm Hg.

Perfluorocarbons (PFC) dissolve more oxygen than water, but still lessthan normal blood. To supply the needed amount of oxygen in circulation,patients may require supplemental oxygen. Highly hydrophobic PFCrequires emulsifiers to stabilize the droplet in blood. Theseemulsifiers interact with proteins and emulsifiers found in bloodleading to instability. As a result, large quantities of PFC incirculation in the blood cannot be tolerated. Small amounts of PFCescape from the blood into the lungs where it is vaporized and breathedout. Large amounts of PFC and emulsifier can have a negative effect onlung function.

Crosslinked, polymerized or encapsulated hemoglobin (pHb) basedartificial oxygen carriers (AOC) are late-comers compared withperfluorocarbon based AOCs described in previous paragraphs, and areattracting increasing attention because their oxygen deliverycharacteristics are similar to that of the red blood cells (hereinafterreferred to as RBC).

Polymeric hemoglobins (pHb) bind O₂ and CO₂, with a binding mechanismmuch like that of red blood cells (RBC), but even a small quantity ofunpolymerized Hb left in the circulation can become very toxic. As anartificial oxygen carrier (AOC), a large amount of pHb needs to beinjected into a person. Premature breakdown can increase the risk oftoxicity, and such a large amount can overtax the body's natural removalprocesses. Polymerized Hb remains costly. Animal sources of Hb run therisk of transferring, among other things prion-based diseases.Recombinant Hb is a promising approach. It requires high qualityseparation and purification procedures, that add to the cost.

While both polymeric hemoglobins (Hb) and perfluorocarbons (PFC) basedAOC products deliver oxygen in significant quantities to cells andtissue, their side effects, such as nitric oxide relatedvasoconstriction, stroke, cardiac arrest, flu-like symptoms and longterm chemical toxicity, have forced the termination of all the clinicaltrials in the U.S. An all out effort to reduce the toxicity ofrelatively large quantity of AOC injected into a body by metabolicdecompositions has failed.

In view of the many problems experienced with artificial blood productsand particulate carriers intended for the controlled delivery ofbiologically active substances within the body, particulate artificialoxygen carriers (AOC) have been developed that minimize the abovedescribed problems in the prior art with non-particulate AOCs. Theparticulate AOCs are designed to be continually circulated in a closedloop fluid circulation system, are less subject to turbulent breakup,chemical decomposition, or accumulation of debris, and are capable ofexchange of small ions and gases.

However, while particulate AOC artificial oxygen carriers minimize theproblems of earlier AOCs that are described above, they break down intime in the blood so there is a need in the art for a way to remove themfrom the body after they have served their purpose as an artificialoxygen carrier.

SUMMARY OF THE INVENTION

The need in the prior art described in the previous paragraph issatisfied by the present invention. To satisfy the above listed need inthe prior art the present invention is a specialized centrifugal rotorthat utilizes density gradient separation to efficiently removeparticulate artificial oxygen carriers (hereinafter referred to asretrievable AOCs or rAOC) from blood or other biofluids. In addition,the rAOC is retrieved from a patients system as soon as its medicalpurpose is accomplished in order to alleviate the physiological stresson already compromised patients.

With the present invention the particulate rAOCs can be retrieved at anydesired time using continuous flow separation employing density-gradientcentrifugation, which may be supplemented with magnetic fields, affinityfiltration or other methods, without suffering damage, or inflictingdamage on other materials that may already be present in the flowingfluid.

Other applications for the present invention include removal andconcentration of metastatic cancer cells from circulating blood,retrieval of low copy mammalian, bacterial or virus cells, and tissueand organ imaging. Depending on the application, the specific designrequirement of these materials in terms of their size and compositionmay vary, but common to all of them are the properties summarizedearlier, and the tailored ability for continuous retrieval fromcirculating fluids using the methods listed in the previous paragraph.

To remove the carrier particles from the blood one or more of thefollowing continuous flow separation methods may be used: (a)centrifugation, (b) magnetic fields, and/or (c) affinity filtrationwithout suffering damage or inflicting damage on other materials thatmay already be present in the flowing fluid. It is contemplated thatparticulate rAOCs be removed from the bloodstream as soon as possibleafter they have performed their function, but prior to degradation ofthe particulate rAOCs, and subsequent development of detrimental sideeffects.

To meet the criteria for retrievability of the above describedparticulate rAOC particles of the present invention from blood duringtheir use, the particulate material must be submicron sized (50 nm-700nm) hollow particles filled with a high density perfluorocarbon (PFC)and/or a poly hemoglobin (pHb) liquid. The hollow particles have one ortwo rigid reinforcing shells. The exterior surface of these particulateshells are coated with molecules containing exposed functional groups(COOH, NH₂, SH etc.) convenient for the crosslinking of either more thanone particle, or proteins like antibodies, cell receptor targets,polyhemoglobin, hemoglobin etc.

The single shell coated emulsion particles (rAOC) of the presentinvention have a higher density than other components of blood such asred blood cells, white blood cells and plasma. Accordingly, centrifugalforces may be utilized to separate the particles from other bloodcomponents, but density gradient is used rather than a sedimentationvelocity method as in the prior art. In the prior art red blood cellsare the furthest moving particles in a centrifugal field, but with thepresent invention the novel AOC is the furthest moving particles in thecentrifugal field. With the AOC being the furthest moving particles in acentrifugal field they may be separated from all other blood components.

rAOCs in the blood have a higher density than the blood and areseparated therefrom by continuous flow density gradient centrifugationthat utilizes the higher density of the rAOC particles to accomplishtheir separation. Affinity filtration may also be used to separate therAOC nano or sub-nano size particles from the blood.

In addition, paramagnetic materials may be added to the higher densityPFC in each nanoparticle, and the magnetic susceptibility is used forthe retrieval of the polymerized hemoglobin. The flowing liquidcontaining paramagnetic and diamagnetic materials (the natural bloodcomponent) must be exposed to a magnetic field during the centrifugalseparation so that they will deviate in the direction of the flow ofparticles with paramagnetic materials away from the diamagneticparticles, thus making it possible to separate and collect both types ofparticles.

Description of the Drawing

The invention will be better understood upon reading the followingDetailed description in conjunction with the drawings in which:

FIG. 1 is a perspective view of the novel centrifuge that utilizesdensity gradient separation to efficiently remove particulate artificialoxygen carriers from blood or other biofluids;

FIG. 2 is a top view of the novel centrifuge that better shows the novelrotor used in the centrifuge;

FIG. 3 is a linear graphical representation of the novel rotor of thecentrifuge;

FIG. 4 is a block diagram of the circuits required for operation of thenovel centrifuge that utilizes density gradient separation toefficiently remove particulate artificial oxygen carriers from blood orother biofluids;

FIGS. 5A and 5B are transmission electron microscope images of submicronsized blood substitutes optimized for use with the described invention;

FIG. 6 is a cross sectional diagram showing how a single shelled rAOC isconstructed; and

FIG. 7 is a cross sectional representation of a double shelled, dualcore oxygen carrier (DCOC) that wraps a PFC emulsion core wrapped with afirst shell on the outside of which is PolyHB that is wrapped with asecond shell; and

DETAILED DESCRIPTION

Prior art coated particulate carriers intended for the controlleddelivery of biologically active or medicinal substances within the body,or to serve as artificial oxygen carriers (AOC), break down in time inthe blood so there is a need in the art for a way to remove them fromthe body after they have served their purpose. Hereinafter, only AOC arespecifically mentioned but the teaching also applies to particulatecarriers intended for the controlled delivery of biologically active ormedicinal substances within the body.

To meet the criteria for coated/particulate artificial oxygen carriersthat can be temporarily substituted for blood, and for theretrievability of such coated AOCs (hereinafter referred to only asretrievable rAOC) from blood using the present invention, the rAOCsdescribed herein are particulates having shells 12 (see FIGS. 5A and 5B)that must be submicron sized (50-1000 nm) hollow particles around a highdensity perfluorocarbon (PFC) emulsified nanoparticle. The reinforcingshell 12 is rigid and consists of a combination of lipids and inorganicmaterials like calcium phosphate, silicate, or biocompatible organicpolymers such as, but not exclusively: polycaprolactone, polylacticacid, polyglycolic acid, polyethylene oxide, chitosan or chondroitin.The rAOCs nanoemulsion core particles 11 are denser than blood and thehigher density is used to retrieve them from blood using a specialcentrifuge. Such shelled rAOCs are shown in and described very brieflywith reference to FIGS. 5, 6, and 7.

Simply, the novel means of the present invention for removing such rAOCsfrom blood comprises having a novel centrifuge rotor 24 that creates adensity gradient that separates the rAOCS from the blood. In the priorart separation of mixed components is based sedimentation velocity. Thisis possible because the density of rAOC is 1.98 g/ml, while the densityof most of the blood components are only slightly over 1.0 g/ml. Amixture of blood and rAOCs withdrawn from the body are input to aspecific point in the centrifuge where the rotation of the centrifugerotor 24 causes the blood to flow in one direction and the rAOCs to flowin the opposite direction, and they are both removed from thecentrifuge. Before the separated rAOCs are retrieved a sample of therAOC flow is removed from the centrifuge and input to a red blood cell(RBC) sensor which looks for any red blood cells. If any red blood cellsare detected electronics of the system adjusts the speed of the pumpsinputting and removing the RBC and rAOC from the centrifuge until no RBCare detected in the rAOCs to be removed from the centrifuge. Inaddition, the rotational speed of the novel rotor inside the centrifugemay also be adjusted. This is shown in and described hereinafter ingreater detail with reference to FIG. 4.

FIG. 1 is a perspective view of the novel centrifuge rotor 24 thatutilizes density gradient separation to efficiently remove particulateartificial oxygen carriers (rAOC) from blood (RBC) or other biofluids.The case of the centrifuge and input and output ports therethrough arenot shown in FIG. 1 to make the drawing simpler so the invention can bebetter understood. Rotor 24 comprises a circular rotor base 25 that ismounted on an axis 27 to a motor driven shaft (not shown). As shown inFIG. 1 rotor base 25 is rotated in a counter clockwise direction for therotor 24 configuration shown and described herein. This direction isimportant, based on the arrangement of rotor elements 26 a and 26 b andtheir position on rotor base 25, to create a density based gradient thatseparates RBC (output at port 29) from the rAOC (output at port 28) froma mixture of RBC and rAOC that is input to the centrifuge at port 31.Distances d3, d4 and dr are shown in all of FIGS. 1, 2 and 3 to betterunderstand how the Figures relate to each other. The thickness of rotor26 a,26 b is 0.5 cm, the width is 2 cm, and the length is 15 cm, and thevolume of the rotor will be only 15 ml.

Rotor 24 is made up of two curved elements 26 a and 26 b that are joinedtogether to form a curved element 26 a,26 b that is orientedperpendicular to rotor base 25. The curvature of element 26 b isslightly larger than the curvature of element 26 a, and curved compositeelement 26 a,26 b is offset on rotor base 25 as may be seen in FIG. 1,but is better seen in the top view of FIG. 2. In FIG. 1 the far left endand the far right end of curved element 26 a,26 b curve outward a smallamount to direct the flow of separated whole blood to output port 29 andto direct the separated/retrieved rAOC to output port 28 where they exitthe centrifuge via their respective ports 28, 29 (not shown) through thecase wall (not shown) of the centrifuge. The different curvatures ofelements 26 a and 26 b and the position of the composite curved element26 a,26 b on rotor base 25 create differing distances d3, d4 and dr inFIG. 1 where d4>dr>d3. These distances are shown in FIGS. 1, 2 and 3 tohelp understand rotor 24 in all the Figures. As shown in FIGS. 1, 2 and3 a mixture of whole blood (RBC) and AOCs is typically extracted from abody (not shown) and is input to the centrifuge at input port 31. Asmentioned above the length of rotor 26 a,26 b is 15 cm but theseparation capacity per unit time could be increased by enlarging thewidth of the rotor 26 a,26 b to greater than 2 cm. In an alternativeembodiment of the invention the curvatures of rotor segments 26 a and 26b may be the same.

FIG. 2 is a top view of the novel rotor 24 used in a centrifuge. Aspreviously mentioned the different curvatures of rotor elements 26 a and26 b and the offset of composite rotor element 26 a,26 b on rotor base25 are best seen in FIG. 2. More particularly, rotor 26 a,26 b beingbelt shaped in the general shape of an ellipsoid with overlapping ends.With rotor 26 a,26 b being off centered on base 25 regions of high,medium and low centrifugal force are created depending on the distancesfrom the axis of rotation 27. As previously mentioned the far left endand the far right end of curved composite element 26 a,26 b curveoutward a small amount to direct the flow of separated whole blood (RBC)to output port 29 and to direct the separated/retrieved rAOC to outputport 28 where they exit the centrifuge via their respective ports 28, 29(not shown) through the case wall (not shown) of the centrifuge. Thecurvature of composite rotor element 26 a,26 b and its position on rotorbase 25 is best seen in this Figure. Input 31 where the compositemixture of RBC and rAOC is input to the centrifuge is offset from thejunction of rotor elements 26 a and 28 b and is closer to rAOC outputport 28 by a circumferential distance “x” as shown. The reason for thisis described further in this Detailed Description. The other input andoutput ports have been previously described with reference to FIG. 1 sothe description is not repeated here. While two rotor segments are shownin FIGS. 1 and 2, in alternative embodiments of the invention there maybe more than two rotor segments.

FIG. 3 is a linear graphical representation of the novel rotor 24 of thecentrifuge. This Figure shows how the distance between the face ofcomposite rotor elements 26 a,26 b and the axis of rotation 27 of rotor24 changes. Thus, the magnitude of centrifugal force at differentregions of rotor 24 are depicted by the distance from the axis ofrotation 27, which is stretched and shown as the dotted line at the topof FIG. 2. The distances d3, d4 and dr are shown in all of FIGS. 1, 2and 3 to better understand how the Figures relate to each other. Therate of change in distance is basically linear except where rotorelement 26 a meets rotor element 26 b. This is due to the fact thecurvature of element 26 a is different than the curvature of element 26b. In alternative embodiments of the invention the rate of change indistance may be uniform, and in another alternative embodiment the rateof change may be non-linear. Distances d3, d4 and dr between the face ofrotor element 26 a,26 b and axis 27 are shown to link FIG. 3 with FIGS.1 and 2. The input port 31 and output ports 28, 29 and 30 and theirrelative position with respect to the linear depiction of rotor 24 isshown.

Whole blood including rAOCs obtained from a person who is connected in aclosed loop system with a density gradient centrifuge is input to thecentrifuge at input port 31. The whole blood is separated from the rAOCbecause the density of the rAOCs is greater than the density of thewhole blood and any of its individual components. The whole blood isoutput at output port 29 and is returned to the person from whom theblood and rAOCs was withdrawn. The rAOCs are output at port 28 andstored for future use or disposal. In addition, at a particular locationnear where the rAOCs exit the centrifuge via rAOC output port 28, asmall sample is removed from the density gradient centrifuge and exitsthe centrifuge at monitor output port 30. The sample is input to a redblood cell sensor 32 of a control circuit 38 to be checked for thepresence of any remaining red blood cells (RBC) with the rAOCs about toexit the centrifuge. This is better shown in and described withreference to FIG. 4. If any RBC are detected control circuit 38 adjuststhe speed of the blood and rAOC pumps 36 and 37 that are part of circuit38 to permit the centrifuge to fully separate any remaining RBC from therAOC before the rAOC reaches monitor output port 30. This feedbackoperation assures that only rAOCs exit rAOC output port 28.

The centrifugal field generated in the density gradient centrifuge asnovel rotor 24 turns about its axis 27 (FIGS. 1 and 2) creates a densitygradient field that changes between output ports 28 and 29. Depending onthe shape of rotor elements 26 a and 26 b, how they are joined, and howthey are positioned on rotor base 25 this density field may changeuniformly or it may non-linearly. The result is that the lower densitywhole blood fraction is separated from the higher density rAOC fraction.In an alternative embodiment another output port may be added somewherebetween output ports 28 and 29 to separate intermediate densityfractions of blood. The separated whole blood and rAOC are withdrawnthrough their respective output ports as previously described. The wholeblood collected may be subjected to further fractionation. For example,further fractionation may be used to separate platelets and white bloodcells from the whole blood in a manner known in the art.

More particularly as novel rotor 24 turns the density gradient field itcreates causes the less dense, faster moving fractions of whole blood tomove toward whole blood output port 29 and the more dense rAOC, however,migrate toward an area of the chamber having the greatest centrifugalforce. By selecting the proper fluid in flow and out flow rates throughthe centrifuge, the physical dimensions of the rotor, and the speed ofrotation of the rotor in the centrifuge, faster moving cells and slowermoving cells may be separately extracted from the separation chamber andsubsequently collected. In this manner, white blood cells and plateletsmay be separated and subsequently collected in separate collectreservoirs. Therefore, the combination of density centrifugation andcentrifugal elutriation provides methods of separating blood componentsbased on both density and sedimentation velocity properties.

The basic design of the centrifuge rotor 26 a,26 b is a belt shapedsemicircular rotor placed slightly off-centered from the axis ofrotation as shown in FIGS. 1 and 2. FIG. 1 is a three dimensional viewof the rotor 26 a,26 b on the spinning rotor base 25, and FIG. 2 is atop view of rotor 26 a,26 b on the spinning rotor base 25. In FIG. 3 therotor 26 a,26 b is shown stretched out in a linear configuration to helpshow the location of the rotor on rotor base 25 with respect to axis ofrotation 27.

The semicircular rotor 26 a,26 b consists of two curved segments 26 aand 26 b, one segment (26 b) slightly more distanced from the axis ofrotation 27 than the other segment (26 a) and therefore experiencinghigher centrifugal force, while the other segment (26 a) is closer tothe axis of rotation and therefore experiences less centrifugal forcethan segment (26 b). A mixture of the blood and high-density particles(rAOC) enter the outer wall of the higher centrifugal force segment 26 bas indicated as “Whole blood and rAOC input 31) in FIGS. 1, 2 and 3.

With reference to FIG. 3, as the centrifugation begins the rAOC of theinput mixture 31 remain at the wall of the furthest out rotor segment 26b, as it is the most dense material and moves towards the highercentrifugal field. This is to the right in FIG. 3 and the output isindicated as “Flow of rAOC F_(r)”. In FIGS. 1 and 2 this is clockwiseand the output is indicated as “rAOC output 28”. All the bloodcomponents move toward the left in FIG. 3 toward closer rotor segment 26a because their densities are smaller and they essentially float on topof the rAOC. In FIGS. 1 and 2 this is counterclockwise and the bloodcomponents output is indicated as “Whole blood output 29”.

More particularly, as the blood and rAOC continue to be injected intorotor 26 a, 26 b at input 31 (shown in FIGS. 1-3), the blood componentsmove towards the lower centrifugal field while the rAOC move to thehigher centrifugal field. The thickness of belt shaped rotor 24 is only5 mm. The separation of the rAOC and blood is carried out very quicklyand form layers based are density of the particles. With separationbeing accomplished quickly it is possible maintain the rate of rAOC andblood inflow sufficiently fast to make the process “continuous-flowdensity separation”. As mentioned above the rAOC leave the rotor atoutput 28 at the end of highest centrifugal force, while the bloodcomponents move leave the rotor at output 29 at the end of lowestcentrifugal force. The semicircular rotor has a small offset, bend andprotrusion near the junction of segments 26 a and 26 b to make theseparation of rAOC from the blood complete. In FIGS. 1, 2 and 3 thisindicated by the number 40, but offset 40 is best seen in FIGS. 2 and 3.More specifically, it is possible to enhance the change of centrifugalforce by creating a protrusion at the site where distinctive separationof two layers is made, since their sedimentation coefficients arepredominantly a function of (1−ρ/δ), the particulates will be positionedclose to the outer wall of the rotor when the density equilibrium isestablished.

Near at the exit port 28 of the rAOC, there is a monitor output port 30,from which small samples are taken of the rAOC flowing toward its output28 to test the purity of the rAOC. The testing of the rAOC is shown inand described with reference to FIG. 4. The purity of the rAOC mightchange slowly over time during centrifugal retrieval of the rAOC so therelative flow rates of pumps 36 and 37 must be adjusted to maintain thepurity of the rAOC output at its port 28. The addition of all out-flowsof the rAOC and blood should equal to the inflow of the blood and rAOC,i.e. Fbr=Fr+Fm+Fb.

In FIG. 4 is a block diagram of circuits required for successfuloperation of the novel centrifuge that utilizes density gradientseparation to efficiently remove particulate artificial oxygen carriers(rAOC) from blood or other biofluids. The circuits first comprise a redblood cell (RBC) sensor 32 that receives the previously mentioned sampleoutput from the centrifuge at monitor output 30. The concentration ofany contaminating low density RBC in the sample taken at output 30 isdetected spectrophotometrically. The output from RBC sensor 32 isamplified by amplifier 33 and is then input to two logic circuits 34 and35. Circuits 34 and 35 are programmed to respond to any output fromsensor 32 to provide output signals that will change the operation ofpumps 36 and 37 which thereby can change either or both of the flow rateof lower density blood flowing out at blood output 29 and higher densityrAOC flowing out at blood output 28. In addition, there can be aprogrammed logic circuit 38 that responds to the output from sensor 32and, in cooperation with logic circuits 34 and 35, provides and outputat 39 to the motor that rotates rotor 24 to change its rotational speed.

FIGS. 5 A&B shows typical electron microscope pictures of the shelledrAOC particles 11. The shells 12 of these novel rAOC particles 11 arecoated with molecules containing exposed functional groups (COOH, NH₂,SH etc.) convenient for the crosslinking of either more than oneparticle, or proteins like antibodies, cell receptor targets,polyhemoglobin, hemoglobin etc. Outer ring or shell 12 is a gas permeantcalcium phosphate or polymer coating, while the interior is an oxygencarrying center containing a hemoglobin (HB) 13 nanoparticle and/or aperfluorocarbon (PFC) 14 nanoparticle.

Very briefly, single shell rAOCs 11 are made as follows. Nanoemulsionparticles 13 are made from a mixture of perfluorooctylbromide (PFOB) 21,1,2-dioleoyl-sn-glycero-phosphate (DOPA)22 and water, preferably by astirring process, but other methods known in the art may be utilized.

The outer surface of the perfluorooctylbromide (PFOB) nanoparticles 11has a surface of 1,2-dioleoyl-sn-glycero-phosphate (DOPA) 22 surroundinga nanomulsion particle 21. The uncoated (non-mineralized) nanoemulsionparticles 13 have a negatively charged surface of PO₃ ⁻ created by usingphosphatidic acid to stabilize the nanoemulsion particles. Since thesynthesis of nanoemulsion particles takes place under basic conditions,the surface charge density of the nanoemulsion is quite high with zetapotentials nearing −50 mV.

To coat the negatively charged nanoemulsions particles 13 they may bemixed with 2:00 μl of 0.1 M phosphoric acid solution. Next, a CaCl₂solution is added followed by a CEPA solution to coat the nanoemulsionparticles and arrest further calcium phosphate deposition. In thisprocess positively charged calcium ions from the phosphoric acid areattracted to the negatively charged PO₃ ⁻ on the surface of thenanoemulsion particles 13 (DOPA) as shown in FIG. 6. The accumulation ofcalcium ions at the periphery of the nanoemulsion particles increasesthe local concentration past the stability point for calcium phosphateprecipitation resulting in precipitation of calcium phosphate onto thenanoemulsion particles to form a shell. The finished shelled, particlesfunction well as oxygen carriers in blood.

A second shell and second oxygen carrier may be added as shown in FIG.7. First, Polylysine/Hb is deposited layer by layer onto the negativelycharged carboxylated surface of the first shell made as described above.Then a mixture of perfluorocarbon (PFC) and Polyhemoglobin (PolyHB) iscoated over the first shell and the same previously described method isused to place a second shell over the PFC and PolyHB. The second shellmakes the rAOC particles tougher and even better able to withstand beingretrieved from circulating blood using the continuous flow densitygradient separation technique described above. The finished shelled,particles function well as oxygen carriers in blood.

The novel density gradient separation technique taught and claimedherein may be used to separate other mixtures of substances havingdifferent densities. It may be used to separate and remove metastaticcancer cells from circulating blood. It may also be used for retrievalof low copy mammalian, bacterial or virus cells from blood. It may alsobe used to remove materials added to blood to enhance tissue and organimaging. Depending on the application, the specific design requirementof these materials in terms of their size and composition may vary, butcommon to all of them are the properties summarized earlier, and thetailored ability for continuous retrieval from circulating fluids.

While what has been described herein is the preferred embodiment of theinvention it will be understood by those skilled in the art thatnumerous changes may be made without departing from the spirit and scopeof the invention.

1. A rotor for a centrifuge used to separate components having differentdensities from a mixture of the components, the rotor comprising: arotor base having a central axis and the rotor base is rotated about thecentral axis when the centrifuge is in use; a first rotor element thatis curved and is attached to and has an orientation extending away fromthe rotor base, the first rotor element having a first end and a secondend; and a second rotor element that is curved and is attached to andhas an orientation extending away from the rotor base, the second rotorelement having a first end and a second end, the second end of the firstrotor element being connected to the first end of the second rotorelement to form a composite rotor element; wherein the composite rotorelement is positioned on the rotor base so that the first end of thefirst rotor element and the second end of the second end of the secondrotor element are at different distances from the central axis.
 2. Thecentrifuge rotor of claim 1 further comprising: a centrifuge housing inwhich the composite rotor element on the rotor base is mounted and isrotated; a first output port through the sidewall of the centrifugehousing for removing a first component of the mixture of componentsinput to the centrifuge housing; a second output port through thesidewall of the centrifuge housing for removing a second component ofthe mixture of components input to the centrifuge housing, the spacingbetween the first and second output ports being substantially the samespacing as the spacing between the first end of the first rotor elementand the second end of the second rotor element; an input port throughthe sidewall of the centrifuge housing through which the mixture ofcomponents is input to the centrifuge housing, said input port beingcloser to the second end of the second rotor element than to the firstend of the second rotor element which is connected to the second end ofthe first rotor element to form the composite rotor element.
 3. Thecentrifuge rotor of claim 2 wherein when the rotor base with compositerotor element mounted thereon is rotated inside the centrifuge housingthe orientation of the composite rotor element on the rotor base createsa density gradient that separates two components of the mixture ofcomponents that is input to the centrifuge housing, where the twocomponents have different densities, and a first of the two componentsmoves in a first direction inside the centrifuge housing and is removedfrom the centrifuge housing at the first output port while a second ofthe two components moves in a second, opposite direction inside thecentrifuge housing and is removed from the centrifuge housing at thesecond output port.
 4. The centrifuge rotor of claim 3 furthercomprising: a monitor port through the sidewall of the centrifugehousing, the monitor port being closer to the second output port at thesecond end of the second rotor element than the input port is, themonitor port being used to extract a sample of the second of the twocomponents moving toward the second output port, the sample being usedto determine if the first of the two components has been separated fromthe second component.
 5. The centrifuge rotor of claim 4 furthercomprising: an outwardly extending end at the first end of the firstrotor segment and at the second end of the second rotor segment, whereinas the rotor turns inside the centrifuge housing these two ends create apressure pushing the first component of the mixture of components towardthe first output port and pushing the second component of the mixture ofcomponents toward the second output port.
 6. The centrifuge rotor ofclaim 5 further comprising: a sensor connected to the monitor outputport to monitor the sample of the second of the two components movingtoward the second output port and extracted at the monitor port for thepresence of any of the first of the two components, the sensorgenerating an output signal if any of the first of the two components ispresent; and electronics receiving the output signal from the sensor,the electronics causing a change in the rate at which the first of thetwo components is removed from the centrifuge at the first output port,and changing the rate at which the second of the two components isremoved from the centrifuge at the second output port to eliminate thepresence of any of the first of the two components in the sample takenat the monitor output port, thus assuring there is none of the first ofthe two components present with the second of the two components exitingthe centrifuge at the second output port.
 7. The centrifuge rotor ofclaim 6 wherein the electronics also causes a change in the rate atwhich the mixture of components is input to the centrifuge housing toassure there is none of the first of the two components present with thesecond of the two components exiting the centrifuge at the second outputport.
 8. The centrifuge rotor of claim 2 further comprising: a monitorport through the sidewall of the centrifuge housing, the monitor portbeing closer to the second output port at the second end of the secondrotor element than the input port is, the monitor port being used toextract a sample of the second of the two components moving toward thesecond output port, the sample being used to determine if the first ofthe two components has been separated from the second component.
 9. Thecentrifuge rotor of claim 8 further comprising: an outwardly extendingend at the first end of the first rotor segment and at the second end ofthe second rotor segment, wherein as the rotor turns inside thecentrifuge housing these two ends create a pressure pushing the firstcomponent of the mixture of components toward the first output port andthe second component of the mixture of components toward the secondoutput port.
 10. The centrifuge rotor of claim 9 wherein when the rotorbase with composite rotor element mounted thereon is rotated inside thecentrifuge housing the orientation of the composite rotor element on therotor base creates a density gradient that separates two components ofthe mixture of components that is input to the centrifuge housing, wherethe two components have different densities, and a first of the twocomponents moves in a first direction inside the centrifuge housing andis removed from the centrifuge housing at the first output port while asecond of the two components moves in a second, opposite directioninside the centrifuge housing and is removed from the centrifuge housingat the second output port.
 11. The centrifuge rotor of claim 4 furthercomprising: a sensor connected to the monitor output port to monitor thesample of the second of the two components moving toward the secondoutput port and extracted at the monitor port for the presence of any ofthe first of the two components, the sensor generating an output signalif any of the first of the two components is present; and electronicsreceiving the output signal from the sensor, the electronics causing achange in the rate at which the first of the two components is removedfrom the centrifuge at the first output port, and changing the rate atwhich the second of the two components is removed from the centrifuge atthe second output port to eliminate the presence of any of the first ofthe two components in the sample taken at the monitor output port, thusassuring there is none of the first of the two components present withthe second of the two components exiting the centrifuge at the secondoutput port.
 12. The centrifuge rotor of claim 11 wherein theelectronics also causes a change in the rate at which the mixture ofcomponents is input to the centrifuge housing to assure there is none ofthe first of the two components present with the second of the twocomponents exiting the centrifuge at the second output port.
 13. Thecentrifuge rotor of claim 12 wherein when the rotor base with compositerotor element mounted thereon is rotated inside the centrifuge housingthe orientation of the composite rotor element on the rotor base createsa density gradient that separates two components of the mixture ofcomponents that is input to the centrifuge housing, where the twocomponents have different densities, and a first of the two componentsmoves in a first direction inside the centrifuge housing and is removedfrom the centrifuge housing at the first output port while a second ofthe two components moves in a second, opposite direction inside thecentrifuge housing and is removed from the centrifuge housing at thesecond output port.
 14. A rotor for a centrifuge used to separate wholeblood from other artificial blood having a density higher than any ofthe components of the whole blood, the rotor comprising: a rotor basehaving a central axis and the rotor base is rotated about the centralaxis when the centrifuge is in use; a first rotor element that is curvedand is attached to and has an orientation extending away from the rotorbase, the first rotor element having a first end and a second end; and asecond rotor element that is curved and is attached to and has anorientation extending away from the rotor base, the second rotor elementhaving a first end and a second end, the second end of the first rotorelement being connected to the first end of the second rotor element toform a composite rotor element; wherein the composite rotor element ispositioned on the rotor base so that the first end of the first rotorelement and the second end of the second end of the second rotor elementare at different distances from the central axis.
 15. The centrifugerotor of claim 14 further comprising: a centrifuge housing in which thecomposite rotor element on the rotor base is mounted and is rotated; afirst output port through the sidewall of the centrifuge housing forremoving the whole blood from the artificial blood input to thecentrifuge housing; a second output port through the sidewall of thecentrifuge housing for removing the higher density artificial bloodinput to the centrifuge housing along with the whole blood, the spacingbetween the first and second output ports being substantially the samespacing as the spacing between the first end of the first rotor elementand the second end of the second rotor element; an input port throughthe sidewall of the centrifuge housing through which the mixture ofwhole blood and artificial blood is input to the centrifuge housing,said input port being closer to the second end of the second rotorelement than to the first end of the second rotor element which isconnected to the second end of the first rotor element to form thecomposite rotor element.
 16. The centrifuge rotor of claim 15 whereinwhen the rotor base with composite rotor element mounted thereon isrotated inside the centrifuge housing the orientation of the compositerotor element on the rotor base creates a density gradient thatseparates the whole blood from the artificial blood where the componentsof the whole blood have a lower density than the artificial blood, and afirst of the whole blood moves inside the centrifuge housing toward andis removed from the centrifuge housing at the first output port whilethe artificial blood moves inside the centrifuge housing toward and isremoved from the centrifuge housing at the second output port.
 17. Thecentrifuge rotor of claim 16 further comprising: a monitor port throughthe sidewall of the centrifuge housing, the monitor port being closer tothe second output port at the second end of the second rotor elementthan the input port is, the monitor port being used to extract a sampleof the artificial blood moving toward the second output port, the samplebeing used to determine if the whole blood has been completely separatedfrom the artificial blood.
 18. The centrifuge rotor of claim 17 furthercomprising: an outwardly extending end at the first end of the firstrotor segment and at the second end of the second rotor segment, whereinas the rotor turns inside the centrifuge housing these two ends create apressure pushing the whole blood toward the first output port and theartificial blood toward the second output port.
 19. The centrifuge rotorof claim 18 further comprising: a sensor connected to the monitor outputport to monitor the sample of the artificial blood moving toward thesecond output port and extracted at the monitor port to test for thepresence of any whole blood components, the sensor generating an outputsignal if any of the first of the two components is present; andelectronics receiving the output signal from the sensor, the electronicscausing a change in the rate at which the first of the two components isremoved from the centrifuge at the first output port, and changing therate at which the second of the two components is removed from thecentrifuge at the second output port to eliminate the presence of any ofthe first of the two components in the sample taken at the monitoroutput port, thus assuring there is none of the first of the twocomponents present with the second of the two components exiting thecentrifuge at the second output port.
 20. The centrifuge rotor of claim19 wherein the electronics also causes a change in the rate at which themixture of whole blood and artificial blood is input to the centrifugehousing to assure there is none of the whole blood components presentwith the artificial blood exiting the centrifuge at the second outputport.